DNA STRUCTURE AND DYNAMICS IN BAMHI-DNA INTERACTIONS

蛋白质对特定DNA序列的识别在许多生物功能中起着重要作用。结构研究表明，这种特异性识别通常涉及DNA结构的扭曲，以适应蛋白质结合。出现了两种模式的机制，这种具体的承认。其中之一，蛋白质通过重新排列自身和弯曲DNA在识别中发挥积极作用。有人建议，在绑定时释放的自由能足以支付所需的结构扭曲。然而，为了使复合物形成有利，弯曲DNA和/或重排蛋白质所需的自由能必须低。 另一个模型表明，DNA具有内在的灵活性，表现在局部，大幅度动态。这些柔性区域提供了在蛋白质结合时容易变形的软点。在这两个模型中，DNA的弯曲必须是一个低自由能的过程，这表明DNA具有一定的内在柔性。为了验证这一假设，对局部DNA动力学的研究必须伴随结构研究。在接下来的三年里，我们计划使用高分辨率核磁共振研究和固态氘核磁共振研究来探索含有BamHI限制性内切酶结合位点的DNA十二聚体的局部结构变异和动力学。具体地，我们提出了以下程序：1）合成氘标记的DNA亚磷酰胺，用于掺入含有BamHI结合位点的DNA十二聚体中。2)使用固态氘NMR来表征十二聚体中各个核苷酸的骨架和糖部分的局部动力学。3)使用高分辨率NMR提供DNA十二聚体的结构描述。

Recognition of specific DNA sequences by proteins plays an important role in many biological functions. Structural studies have shown that this specific recognition usually involves distortion of the DNA structure to accommodate protein binding. Two models for the mechanism of this specific recognition have emerged. In one, the protein plays an active role in recognition by rearranging itself and bending the DNA. It is suggested that the free energy released upon binding is sufficient to pay for that required for structural distortions. However, in order for complex formation to be favorable, the free energy required to bend the DNA and/or rearrange the protein must be low. Another model suggests that DNA possesses an intrinsic flexibility manifested in local, large amplitude dynamics. These flexible regions provide soft-spots which are easily distorted upon protein binding. In both these models, bending of the DNA must a low free energy process, suggesting that DNA has some inherent flexibility. In order to test this hypothesis, studies of local DNA dynamics must accompany structural investigations. In the next three years we plan to use high-resolution NMR studies and solid-state deuterium NMR studies to explore the local structural variations and dynamics of a DNA dodecamer containing the binding site for the BamHI restriction endonuclease. Specifically we propose the following program l) Synthesize deuterium labeled DNA phosphoramidites for incorporation into a DNA dodecamer containing the BamHI binding site. 2) Use solid-state deuterium NMR to characterize the local dynamics of backbone and sugar moieties of the individual nucleotides in the dodecamer. 3) Use high-resolution NMR to provide a structural description of the DNA dodecamer.